Isolated Polynucleotides and Methods of Promoting a Morphology in a Fungus

ABSTRACT

The invention includes isolated polynucleotide molecules that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention includes a method of enhancing a bioprocess utilizing a fungus. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to a promoter. The polynucleotide sequence is expressed to promote a first morphology. The first morphology of the transformed fungus enhances a bioprocess relative to the bioprocess utilizing a second morphology.

RELATED PATENT DATA

This patent is a divisional of U.S. patent application Ser. No.10/442,017 that was filed May 19, 2003 and which claims benefit ofpriority under 35 U.S.C. §119 to U.S. Provisional Patent Ser. No.60/382,132, which was filed May 20, 2002.

CONTRACTUAL ORIGIN OF THE INVENTION

This invention was made with Government support under contractDE-AC0676RLO-1830, awarded by the U.S. Department of Energy. TheGovernment has certain rights in this invention.

TECHNICAL FIELD

The invention pertains to isolated polynucleotide molecules, recombinantpolynucleotide constructs, and methods of promoting a morphology in afungus.

BACKGROUND OF THE INVENTION

Fungi are becoming increasingly utilized for production of numerouscommercially useful products. A type of fungi known as “filamentous”fungi are currently used for the industrial scale production ofmetabolites such as antibiotics (penicillins and cephalosporins, forexample) and organic acids (citric and fumaric acids for example).Filamentous fungi are additionally useful for the industrial productionof enzymes such as, for example, proteases and lipases.

Utilization of a filamentous fungus species for production of desiredcompounds often involves growing submerged cultures of the fungus.Filamentous fungi can exhibit numerous morphologies in submergedcultures, one of which is the filamentous morphology. When fungi inculture exhibit a filamentous morphology, the filamentous growth canincrease the viscosity of the culture medium. The increased viscositycan affect the mass transfer and aeration properties of the culture, cancause mixing problems in a bioreactor, and can typically be accompaniedby decreased productivity.

Alternatively, “filamentous” fungi can exhibit a pellet morphology. Incontrast to cultures of fungi exhibiting a filamentous morphology, theviscosity of cultures of fungi exhibiting a pellet morphology can berelatively low and can utilize less power for mixing and aeration of theculture. For many products, for example citric acid, itaconic acid,statins, penicillins, and various enzymes, productivity can be enhancedutilizing fungus exhibiting a pellet morphology relative to fungusexhibiting a filamentous morphology. However, at least in certain fungalspecies, production of peptic enzyme or fumaric acid, for example, canbe enhanced by utilizing a fungus exhibiting a filamentous morphology.

It would be desirable to develop methods to promote a desired morphologyin a fungus and to develop methods for influencing or controllingmorphologies exhibited by a fungus in a culture to optimizeproductivity.

SUMMARY OF THE INVENTION

In one aspect, the invention encompasses an isolated polynucleotidemolecule that is differentially expressed in a native fungus exhibitinga pellet morphology relative to the native fungus exhibiting afilamentous morphology.

In one aspect, the invention encompasses a method of enhancing abioprocess utilizing a fungus. A transformed fungus is produced bytransforming a fungus with a recombinant polynucleotide molecule. Therecombinant polynucleotide molecule contains an isolated polynucleotidesequence linked operably to a promoter. A polypeptide encoded by thepolynucleotide sequence is expressed to promote a pellet morphology. Thepellet morphology of the transformed fungus enhances a bioprocessrelative to the bioprocess utilizing a filamentous morphology of thetransformed fungus.

In one aspect, the invention encompasses a method of promoting amorphology of a fungus and enhancing productivity of a bioprocess. Afungus is transformed with an antisense oriented polynucleotide sequencecomplimentary to a gene sequence. A transcription product of thepolynucleotide sequence hybridizes to an mRNA and thereby suppressesexpression of the gene. The gene suppression promotes a morphology andenhances a bioprocess relative to the bioprocess in an alternativefungal morphology.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are described below withreference to the following accompanying drawings.

FIG. 1 shows the results of Northern blot analysis of thetranscriptional level of the native A. niger gene corresponding to theBalu-4 cDNA sequence set fourth in SEQ ID NO.:1. Lanes 1, 2 and 3reflect transcription levels in the pellet morphology. Transcriptionlevels in the filamentous morphology are shown at 20 minutes (lane 4),40 minutes (lane 5) and 120 minutes (lane 6) after inducing thefilamentous morphology.

FIG. 2 shows the alignment and comparison of the predicted amino acidsequence of A. niger Balu-4, SEQ ID NO.:2 (top sequence) and the aminoacid sequence of Emericella nidulans G-protein beta subunit, SEQ IDNO.:3 (bottom sequence).

FIG. 3 shows the results of Northern blot analysis of transcriptionlevels of the native A. niger gene corresponding to the Balu-42 cDNAsequence set forth in SEQ ID NO.:4. Lanes 1, 2 and 3 reflecttranscription levels in the pellet morphology. Transcription levels inthe filamentous morphology are shown at 20 minutes (lane 4), 40 minutes(lane 5) and 120 minutes (lane 6) after inducing the filamentousmorphology.

FIG. 4 shows the results of Northern blot analysis of transcriptionlevels of the native A. niger gene corresponding to the Brsa-25 cDNAsequence set forth in SEQ ID NO.:6. Lanes 1, 2 and 3 reflecttranscription levels in the pellet morphology. Transcription levels inthe filamentous morphology are shown at 20 minutes (lane 4), 40 minutes(lane 5) and 120 minutes (lane 6) after inducing the filamentousmorphology.

FIG. 5 shows the results of Northern blot analysis of transcriptionlevels of the native A. niger gene corresponding to the Brsa-43 cDNAsequence set forth in SEQ ID NO.:8. Lanes 1, 2 and 3 reflecttranscription levels in the pellet morphology. Transcription levels inthe filamentous morphology are shown at 20 minutes (lane 4), 40 minutes(lane 5) and 120 minutes (lane 6) after inducing the filamentousmorphology.

FIG. 6 shows the alignment and comparison of the predicted amino acidsequence of A. niger Brsa-43, SEQ ID NO.:10 (top sequence), and theamino acid sequence of the Homo sapiens lysosomal pepstatin insensitiveprotease, SEQ ID NO. :11 (bottom sequence).

FIG. 7 shows the results of Northern blot analysis of transcriptionlevels of the native A. niger gene corresponding to the Brsa-47 cDNAsequence set forth in SEQ ID NO.:12. Lanes 1, 2 and 3 reflecttranscription levels in the pellet morphology. Transcription levels inthe filamentous morphology are shown at 20 minutes (lane 4), 40 minutes(lane 5) and 120 minutes (lane 6) after inducing the filamentousmorphology.

FIG. 8 shows the alignment and comparison of the predicted amino acidsequence of A. niger Brsa-47, SEQ ID NO.:14 (top sequence), and theamino acid sequence of Sesamum indicum Myo-inositol 1-phosphatesynthase, SEQ ID NO.:15 (bottom sequence).

FIG. 9 shows the results of Northern blot analysis of transcriptionlevels of the native A. niger gene corresponding to the Brsa-109 cDNAsequence set forth in SEQ ID NO.:16. Lanes 1, 2 and 3 reflecttranscription levels in the pellet morphology. Transcription levels inthe filamentous morphology are shown at 20 minutes (lane 4), 40 minutes(lane 5) and 120 minutes (lane 6) after inducing the filamentousmorphology.

FIG. 10 shows the results of Northern blot analysis of transcriptionlevels of the native A. niger gene corresponding to the Brsa-118 cDNAsequence set forth in SEQ ID NO.:18. Lanes 1, 2 and 3 reflecttranscription levels in the pellet morphology. Transcription levels inthe filamentous morphology are shown at 20 minutes (lane 4), 40 minutes(lane 5) and 120 minutes (lane 6) after inducing the filamentousmorphology.

FIG. 11 shows the alignment and comparison of the predicted amino acidsequence of A. niger Brsa-118, SEQ ID NO.:20 (top sequence), and theNeurospora crassa probable hydroxymethylglutaryl-CoA synthase, SEQ IDNO.:21 (bottom sequence).

FIG. 12 shows the results of Northern blot analysis of transcriptionlevels of the native A. niger gene corresponding to the Arsa-7 cDNAsequence set forth in SEQ ID NO.:22. Lanes 1, 2 and 3 reflecttranscription levels in the pellet morphology. Transcription levels inthe filamentous morphology are shown at 20 minutes (lane 4), 40 minutes(lane 5) and 120 minutes (lane 6) after inducing the filamentousmorphology.

FIG. 13 shows the results of Northern blot analysis of transcriptionlevels of the native A. niger gene corresponding to the Arsa-48 cDNAsequence set forth in SEQ ID NO.:24. Lanes 1, 2 and 3 reflecttranscription levels in the pellet morphology. Transcription levels inthe filamentous morphology are shown at 20 minutes (lane 4), 40 minutes(lane 5) and 120 minutes (lane 6) after inducing the filamentousmorphology.

FIG. 14 shows the results of Northern blot analysis of transcriptionlevels of the native A. niger gene corresponding to the A-37 cDNAsequence set forth in SEQ ID NO.:26. Lanes 1, 2 and 3 reflecttranscription levels in the pellet morphology. Transcription levels inthe filamentous morphology are shown at 20 minutes (lane 4), 40 minutes(lane 5) and 120 minutes (lane 6) after inducing the filamentousmorphology.

FIG. 1 5 shows the results of Northern blot analysis of transcriptionlevels of the native A. niger gene corresponding to the A-90 cDNAsequence set forth in SEQ ID NO.:28. Lanes 1, 2 and 3 reflecttranscription levels in the pellet morphology. Transcription levels inthe filamentous morphology are shown at 20 minutes (lane 4), 40 minutes(lane 5) and 120 minutes (lane 6) after inducing the filamentousmorphology.

FIG. 16 shows the results of Northern blot analysis of transcriptionlevels of the native A. niger gene corresponding to the Arsa-43 cDNAsequence set forth in SEQ ID NO.:33. Lanes 1, 2 and 3 reflecttranscription levels in the pellet morphology. Transcription levels inthe filamentous morphology are shown at 20 minutes (lane 4), 40 minutes(lane 5) and 120 minutes (lane 6) after inducing the filamentousmorphology.

FIG. 17 shows the alignment and comparison of the predicted amino acidsequence of A. niger Arsa-43, SEQ ID NO.:34 (top sequence), and theAspergillus nidulans polyubiquitin protein, SEQ ID NO.:35 (bottomsequence).

FIG. 18 shows the results of Northern blot analysis of transcriptionlevels of the native A. niger gene corresponding to the Arsa-10 cDNApartial sequence set forth in SEQ ID NO.:36. Lanes 1, 2 and 3 reflecttranscription levels in the pellet morphology. Transcription levels inthe filamentous morphology are shown at 20 minutes (lane 4), 40 minutes(lane 5) and 120 minutes (lane 6) after inducing the filamentousmorphology.

FIG. 19 shows the results of Northern blot analysis of transcriptionlevels of the native A. niger gene corresponding to the Arsa-27 cDNApartial sequence set forth in SEQ ID NO.:37. Lanes 1, 2 and 3 reflecttranscription levels in the pellet morphology. Transcription levels inthe filamentous morphology are shown at 20 minutes (lane 4), 40 minutes(lane 5) and 120 minutes (lane 6) after inducing the filamentousmorphology.

FIG. 20 shows a comparison of enhanced expression levels in filamentousmorphology (right) relative to the pellet morphology (left) of native A.niger for each of the Balu-4, Brsa-25, Brsa-43, Brsa-47, Brsa-1 09, andBrsa-118 genes.

FIG. 21 shows a comparison of enhanced expression levels in the pelletmorphology (left) relative to filamentous morphology (right) of nativeA. niger for each of the Arsa-7, Arsa-10, Arsa-27, A-27, Arsa-43 andA-90 genes.

FIG. 22 shows the results of Northern blot analysis of transcriptionlevels of the native A. niger genes corresponding to the Balu-4,Balu-42, Brsa-25, Brsa-47, Brsa-109, and Brsa-118 cDNA sequences setforth in SEQ ID NOs.:1, 4, 6, 12, 16 and 18, respectively. Panel (A)shows transcription levels in native A. niger grown in 10 ppb Mn²⁺(pellet morphology) for 14 hr (lane 1), 24 hr (lane 2), 48 hr (lane 3),72 hr (lane 4), 96 hr (lane 5) and 120 hr (lane 6). Panel (B) showstranscription levels in native A. niger grown in 1 000 ppb Mn²⁺(filamentous morphology) for 1 hr (lane 1), 2 hr (lane 2), 24 hr (lane3), 36 hr (lane 4), 72 hr (lane 5) and 108 hr (lane 6).

FIG. 23 shows the results of Northern blot analysis of transcriptionlevels of the native A. niger genes corresponding to the Arsa-7, A-37,Arsa-48, and A-90 cDNA sequences set forth in SEQ ID NOs.:22, 24, 26 and28, respectively. Panel (A) shows transcription levels in native A.niger grown in 10 ppb Mn²⁺ (pellet morphology) for 14 hr (lane 1), 24 hr(lane 2), 48 hr (lane 3), 72 hr (lane 4), 96 hr (lane 5) and 120 hr(lane 6). Panel (B) shows transcription levels in native A. niger grownin 1000 ppb Mn²⁺ (filamentous morphology) for 1 hr (lane 1), 2 hr (lane2), 24 hr (lane 3), 36 hr (lane 4), 72 hr (lane 5) and 108 hr (lane 6).

FIG. 24 is a flowchart diagram illustrating a particular aspect of thepresent invention.

FIG. 25 shows suppression results for A. niger transformed withantisense oriented polynucleotide sequences complimentary to Balu-42(Panel A), Brsa-25 (Panel B) and Brsa-118 (Panel C). Each panel comparesmorphologies of control A. niger (left) and transformed A. niger (right)containing the corresponding antisense DNA construct grown in 15 ppbMn²+medium.

FIG. 26 shows suppression results for A. niger transformed withantisense oriented polynucleotide sequences complimentary to cDNAscorresponding to Arsa-7 (Panel A), A-37 (Panel B) and A-90 (Panel C).Each panel compares morphologies of control A. niger (left) andtransformed A. niger (right) grown in 12 ppb Mn²⁺ medium.

FIG. 27 shows the citric acid production of control A. niger andtransformed A. niger containing antisense polynucleotide sequencecomplimentary to Balu-42 (strain 2805) or complimentary to Brsa-118(strain 2808). Panel (A) shows measured citric acid production forindividual transformation experiments. Panel (B) shows averaged valuesof the data depicted in Panel (A).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention encompasses polynucleotides that can have differentialexpression in a native fungus. For purposes of the present descriptionthe term “expression” of a polynucleotide sequence can refer to thecombined processes of transcription and translation, or can refer to aportion of the combined transcription and translation process. The term“differential expression” can refer to two or more differing levels ofexpression, or can refer to an absence in expression in a first instancerelative to a presence of expression in a second instance.

The invention includes isolated polynucleotide molecules that caninclude a polynucleotide sequence that is differentially expressed indifferent morphologies exhibited by a native fungus. For purposes of thepresent description, the term “native” can refer to an organism that hasnot been genetically manipulated. The term “isolated” can refer to anaturally occurring molecule such as, for example, a polynucleotide or apolypeptide that has been recovered from the organism which produced it,or alternatively can refer to a synthetic molecule.

An isolated polynucleotide molecule according to the present inventioncan comprise a polynucleotide sequence that has an increased expressionin a fungus exhibiting a pellet morphology relative to a lower level oran absence of expression in the filamentous morphology of the fungus.Alternatively, a polynucleotide molecule according to the presentinvention can comprise polynucleotide sequence having an increasedexpression level in a filamentous morphology of a native fungus relativeto a lower level or absence of expression in the pellet morphology.

Isolated polynucleotides encompassed by the present invention can beisolated from any source fungus that is capable of exhibiting afilamentous morphology and a pellet morphology. A source fungus is notlimited to a specific group of fungi and can be a member any of thethree major fungi groups. An exemplary member of the Basidiomycetesgroup is Phanerochaete chrysosporium. Exemplary members of the group ofAscomycetes and Imperfect Fungus include Aspergillus niger, Aspergillusoryzae, Aspergillus terreus, Emericella nidulans, Neurospora crassa,Fusarium oxysporum, Penicillium chrysogenum, and Trichoderma reesei.Exemplary members of the Zygomycetes group include Rhizomucor miehei andRhizopus oryzae.

An exemplary isolated polynucleotide molecule encompassed by the presentinvention can comprise a polynucleotide sequence isolated from A. nigerthat is differentially expressed in the filamentous morphology of nativeA. niger relative to the pellet morphology of native A. niger. Thedifferentially expressed polynucleotide sequence can comprise, forexample, a sequence as set forth in any of SEQ ID NOs.:1, 4, 6, 8, 12,16, 18, 22, 24, 26, 28, 33, 36 and 37, or can comprise a sequencecomplimentary to any of those sequences. Each of the polynucleotidesequences set forth in SEQ ID NOs.:1, 4, 6, 8,12,16, 18, 22, 24, 26, 28,33, 36 and 37, corresponds to the sequence determined from a full-lengthcDNA molecule prepared according to methods discussed below, with SEQ IDNOs.:36 and 37 being partial sequences determined from full length cDNA.It is to be understood that the isolation methods and techniquesdiscussed herein are exemplary and that numerous conventional techniquescan be utilized for producing the isolated polynucleotide molecules ofthe present invention.

Full-length cDNA molecules comprising the polynucleotide sequences setforth in SEQ ID NOs.:1, 4, 6, 8, 12, 16, 18, 22, 24, 26, 28, 33, 36 and37, are obtained from A. niger strain ATCC11414 utilizing suppressionsubtractive hybridization techniques (Diatchenko et al., ProceedingsNational Academy of Science U.S.A. Vol. 93, pp. 6025-6030, 1996), inconjunction with PCR-SELECT™ cDNA subtraction kit (CLONTECH, Palo Alto,Calif.). Two suppression subtractive cDNA libraries are constructed. Afirst cDNA library is constructed utilizing cDNA obtained from A. nigerexhibiting the pellet type morphology as tester and cDNA obtained fromA. niger exhibiting the filamentous morphology as a driver. Thedriver/tester ratio is increased threefold over the ratio suggested bythe subtraction kit manual.

A second suppression subtractive cDNA library is created utilizing cDNAobtained from A. niger exhibiting the filamentous morphology as testerand utilizing cDNA obtained from A. niger exhibiting pellet morphologyas driver. A first cDNA pool is generated from the first library and asecond cDNA pool is generated from the second library. Differentiallyexpressed cDNAs that are specifically present or enhanced in the pelletmorphology are isolated from the first cDNA library by hybridizationutilizing the first cDNA pool as probes and independently hybridizingutilizing the second cDNA pool as probes. Isolation of cDNA that isenhanced or specific to the filamentous morphology of A. niger isachieved by independently hybridizing the second cDNA library utilizingthe first cDNA pool and the second cDNA pool as probes.

The segments of differentially expressed cDNAs that are isolated bysuppression subtractive hybridization are selected for DNA sequencing.Sequencing of the segments is performed utilizing single pass sequencingwith the T7-2 primer. The DNA fragments isolated by the suppressionsubtractive hybridization are used to design pairs of gene specificprimers for utilization in isolating full-length cDNAs.

Full-length cDNA isolation is achieved utilizing the marathon cDNAamplification kit and the ADVANTAGE® cDNA polymerase (CLONTECH, PaloAlto, Calif.). The gene specific primers designed from the suppressionsubtractive hybridization clones are utilized for performing rapidamplification of cDNA ends PCR (RACE-PCR). The sequence of full-lengthcDNAs is determined using conventional automated DNA sequencing methods.

Twelve full-length cDNA clones and two partial-length cDNA clones areproduced and sequenced according to the methods discussed above. Theresulting sequences are presented as follows. The sequence of the Balu-4cDNA is set forth in SEQ ID NO.:1; the sequence of the Balu-42 cDNA isset forth in SEQ ID NO.:4; the sequence of the Brsa-25 cDNA is set forthin SEQ ID NO.:6; the sequence of the Brsa-43 cDNA is set forth in SEQ IDNO.:8; the sequence of the Brsa-47 cDNA is set forth in SEQ ID NO.:12;the sequence of the Brsa-109 cDNA is set forth in SEQ ID NO.:16; thesequence of the Brsa-118 cDNA is set forth in SEQ ID NO.:18; thesequence of the Arsa-7 cDNA is set forth in SEQ ID NO.:22; the sequenceof the Arsa-48 cDNA is set forth in SEQ ID NO.:24; the sequence of theA-37 cDNA is set forth in SEQ ID NO.:26; the sequence of the A-90 cDNAis set forth in SEQ ID NO.:28; the sequence of the Arsa-43 cDNA is setforth in SEQ ID NO.:33; the partial sequence of the Arsa-10 cDNA is setforth in SEQ ID NO.:36; and the partial sequence of the Arsa-27 cDNA isset forth in SEQ ID NO.:37.

The amino acid sequence of each of the fourteen determinedpolynucleotide sequences is predicted utilizing the known genetic code.Homology searches are performed utilizing BLASTP to investigate homologybetween a predicted amino acid sequence and the sequences in the NCBInon-redundant GenBank CDS. All homology searches are conducted utilizinga threshold E value of E=0.005. Accordingly, the results of each BLASThomology search (discussed below) are based upon this initial thresholdvalue.

Northern blot analysis is utilized to analyze the expression levels ofthe genes in native A. niger corresponding to each of the fourteen cDNAclones. The expression of each gene by A. niger exhibiting filamentousmorphology is compared to the expression of the same gene in A. nigerexhibiting the pellet morphology. For expression analysis, A. niger isinitially grown in a culture medium containing less than or equal toabout 12 parts per billion (ppb) Mn²⁺ for 12 hours. After the initial 12hours of growth the culture is divided into two halves, the first halfis maintained at low Mn²⁺ concentration (less than or equal to about 12ppb) and the other half is brought to a final concentration ofapproximately 1000 ppb Mn²⁺ (or in some instances to a finalconcentration of greater than or equal to about 15 ppb Mn²⁺). A. nigercan be extremely sensitive to Mn²⁺ concentration. At Mn²⁺ concentrationsat or below about 12 ppb, native A. niger exhibits the pelletedmorphology, while at Mn²⁺ concentrations higher than about 12 ppb,native A. niger exhibits filamentous morphology. To simplify the presentdescription, the point at which the culture is divided into two halves(after 12 hours of initial growth) can be referred to as time zero(t=0). Additionally, since the addition of Mn²⁺ to a final concentrationof above 12 ppb promotes the filamentous morphology, the addition ofMn²⁺ can be referred to as filament induction.

Culture samples are collected at 20, 40, 60 and 120 minutes after timezero from both the non-induced culture (pellet morphology) and theinduced culture (filamentous morphology). The samples are centrifuged toform culture pellets which are frozen with liquid nitrogen and stored at−80° C. for future total RNA extraction.

Total RNA can be isolated from the frozen culture pellets utilizingconventional methods. After size fractionation of the total RNA sampleby conventional gel electrophoresis techniques and subsequent transferto a blotting membrane, the total RNA samples collected at each timepoint are analyzed using hybridization of probes that are synthesized byrandomly priming the isolated suppression subtractive hybridization cDNAfragments or by randomly priming fragments of full-length cDNA digestedwith restricting endonuclease. Probe synthesis includes incorporation of[³²P]-a-dCTP. Hybridization results of the Northern blots can bevisualized by exposing the blots to x-ray film.

FIG. 1 shows the x-ray film exposure of a Northern blot analysis of theexpression of the A. niger gene corresponding to Balu-4 SEQ ID NO.:1.Increased hybridization is apparent in mRNA samples taken fromfilamentous cultures (lanes 4, 5 and 6) relative to mRNA produced inpellet morphology (lanes 1-3). Fifteen micrograms (μg) of total RNA isused for each lane. The RNA samples utilized are obtained from post t=0pellet cultures at t=20 minutes (lane 1), t=40 minutes (lane 2) andt=120 minutes (lane 3); and from post-induction filamentous cultures att=20 minutes (lane 4), t=40 minutes (lane 5) and t=1 20 minutes (lane6). The total RNA used for each lane and the lane identification foreach of the Northern blots discussed below is the same as that set forthfor FIG. 1. The results shown in FIG. 1 indicate that Balu-4 isdifferentially expressed in native A. niger, with an increased level ofexpression detected in the filamentous morphology.

The predicted amino acid sequence of Balu-4 is set forth in SEQ IDNO.:2. The Balu-4 amino acid sequence is predicted from the Balu-4 cDNAsequence (SEQ ID NO.:1). As shown in FIG. 2, an amino acid sequencehomology search utilizing BLASTP indicates that SEQ ID NO.:2 (topsequence) has a 97% identity with the amino acid sequence of a G-proteinbeta subunit of Emericella nidulans, SEQ ID NO.:3 (bottom sequence).Positions of sequence identity are indicated by the placement of thecorresponding identical amino acid symbol between SEQ ID NO.:2 (top) andSEQ ID NO.:3 (bottom). The symbol “+” shown intermediate SEQ ID NO.:2and SEQ ID NO.:3 indicates a conservative amino acid difference. Forpurposes of the present invention a conservative amino acid differenceor a conservative amino acid substitution can refer to a substitution ofone amino acid by another amino acid with similar chemical properties.Additionally, the term “homology” can, in some instances, refer to anidentical or a conservative amino acid.

The appearance of an open space between corresponding positions in SEQID NO.:2 and SEQ ID NO.:3 in FIG. 2 indicates a non-conservative aminoacid difference between the two aligned sequences. Three sections of SEQID NO.:2 having relatively minimal identity with SEQ ID NO.:3 are setforth as SEQ ID NOs.:30, 31 and 32. SEQ ID NO.:30 corresponds to aminoacids 28-49 of SEQ ID NO.:2. SEQ ID NO.:31 corresponds to amino acids194-209 of SEQ ID NO.:2. SEQ ID NO.:32 corresponds to amino acids260-288 of SEQ ID NO.:2.

FIG. 3 shows the results of Northern blot analysis of the expression ofthe native gene corresponding to Balu-42, SEQ ID NO.:4. The increaseddetection of mRNA corresponding to Balu-42 in the filamentous morphologyindicates that Balu-42 is differentially expressed with increasedexpression in filaments relative to the pellet morphology of native A.niger.

SEQ ID NO.:5 corresponds to the Balu-42 amino acid sequence predictedfrom SEQ ID NO.:4. A BLASTP homology search is unable to identifyhomology between SEQ ID NO.:5 and any sequence in the searched database.

FIG. 4 shows the results of Northern blot analysis of the expression ofthe native gene corresponding to the Brsa-25 cDNA sequence set forth inSEQ ID NO.:6. The results indicate that Brsa-25 is differentiallyexpressed with increased expression in the filamentous morphology ofnative A. niger relative to the pellet morphology.

The predicted amino acid sequence of Brsa-25 SEQ ID NO.:6 is set forthin SEQ ID NO.:7. A BLASTP homology search was unable to identifyhomology between SEQ ID NO.:7 and any sequence in the searched database.

FIG. 5 shows results of the Northern blot analysis of the expression ofthe native gene corresponding to the Brsa-43 cDNA set forth in SEQ IDNO.:8. The Northern blot results indicate that Brsa-43 is differentiallyexpressed with increased expression in the filamentous morphology ofnative A. niger relative to the pellet morphology.

The Brsa-43 amino acid sequence predicted from SEQ ID NO.: 8 is setforth in SEQ ID NO.: 9. SEQ ID NO.:10 corresponds to amino acids 29-594of SEQ ID NO.:9. FIG. 6 shows the BLASTP alignment and comparison ofBrsa-43 SEQ ID NO.:10 (top sequence) which has 31% identity to the aminoacid sequence of human tripeptidyl-peptidase I precursor (lysosomalpepstatin insensitive protease), SEQ ID NO.:11 (bottom sequence).Indication of identity and homology between sequences is as discussedabove with respect to FIG. 2.

FIG. 7 shows the results of Northern blot analysis of the expression ofthe native Brsa-47 gene corresponding to the cDNA sequence set forth inSEQ ID NO.:12. The results indicate that Brsa-47 is differentiallyexpressed; with increased expression levels apparent in the filamentousmorphology relative to the pellet morphology of native A. niger.

The amino acid sequence of Brsa-47 as predicted from SEQ ID NO.:12 isset forth in SEQ ID NO.:13. FIG. 8 shows the BLASTP homology searchresults for SEQ ID NO.:14 (top sequence) which corresponds to aminoacids 26-530 of SEQ ID NO.:13. The BLASTP results indicate that SEQ IDNO.:14 has a 56% identity with the amino acid sequence of Myo-inositol1-phosphate synthase from Sesamum indicum, SEQ ID NO.:15 (bottomsequence).

The results of Northern blot analysis of the expression of the Brsa-109gene in native A. niger corresponding to the cDNA sequence set forth inSEQ ID NO. :16 is shown in FIG. 9. The results indicate that theBrsa-109 gene is differentially expressed, with increased expressiondetected in the filamentous morphology relative to the pelletmorphology.

The Brsa-109 amino acid sequence predicted from SEQ ID NO.:16, is setforth in SEQ ID NO.:17. A BLASTP homology search is unable to identifyhomology between SEQ ID NO.:19 and any sequence in the database.

FIG. 10 shows the results of Northern blot analysis of the expression ofthe Brsa-118 gene in native A. niger corresponding to the cDNA sequenceset forth in SEQ ID NO.:18. The results indicate that the Brsa-118 geneis differentially expressed, with increased expression in thefilamentous morphology relative to the pellet morphology.

The amino acid sequence of Brsa-118 predicted from SEQ ID NO.:18 is setforth in SEQ ID NO.:19. FIG. 11 shows the BLASTP homology search resultsfor Brsa-118. The results show that the predicted amino acid sequence ofBrsa-118, SEQ ID NO.:20 (top sequence), has 66% identity with the aminoacid sequence of probable hydroxymethylglutaryl-CoA synthase fromNeurospora crassa, SEQ ID NO.:21 (bottom sequence).

FIG. 12 shows the results of Northern blot analysis of the expression ofthe Arsa-7 gene in native A. niger corresponding to the cDNA sequenceset forth in SEQ ID NO.:22. The results indicate that the Arsa-7 gene isdifferentially expressed, with increased expression levels in the pelletmorphology relative to expression levels in the filamentous morphology.

The amino acid sequence of Arsa-7 as predicted from SEQ ID NO.: 22 isset forth in SEQ ID NO.:23. BLAST homology search results were unable toidentify any sequences with homology to the predicted amino acidsequence of Arsa-7.

FIG. 13 shows the results of Northern blot analysis and the expressionof the Arsa-48 gene in native A. niger corresponding to the cDNAsequence set forth in SEQ ID NO.:24. The results indicate the Arsa-48gene is differentially expressed, with increased expression levelsoccurring in the pellet morphology relative to the filamentousmorphology.

The Arsa-48 amino acid sequence as predicted from SEQ ID NO.:24, is setforth in SEQ ID NO.:25. A BLASTP homology search was unable to identifyhomology between the Arsa-48 amino acid sequence and any other aminoacid sequence in the searched database.

FIG. 14 shows the results of a Northern blot analysis of the expressionof the A-37 gene in native A. niger corresponding to the cDNA sequenceset forth in SEQ ID NO.:26. The results indicate that the A-37 gene isdifferentially expressed with increased expression occurring in thepellet morphology relative to the expression level detected in thefilamentous morphology.

The A-37 amino acid sequence as predicted from SEQ ID NO.:26, is setforth in SEQ ID NO.:27. The BLASTP homology search was unable to detectany homology between the predicted A-37 amino acid sequence and otheramino acid sequences in the searched database.

FIG. 15 shows the result of Northern blot analysis of the expression ofthe A-90 gene in native A. niger corresponding to the cDNA sequence setforth in SEQ ID NO.:28. The results indicate that A-90 is differentiallyexpressed with an increased expression level occurring in the pelletmorphology relative to the expression level detected in the filamentousmorphology.

The amino acid sequence of A-90 as predicted from SEQ ID NO.:28, is setforth in SEQ ID NO.:29. A BLASTP homology search performed on SEQ IDNO.:29, is unable to detect any homology with any other amino acidsequence in the database.

FIG. 16 shows the results of Northern blot analysis of the expression ofthe Arsa-43 gene in native A. niger corresponding to the cDNA sequenceset forth in SEQ ID NO.:33. The results indicate that the Arsa-43 geneis differentially expressed, with increased expression in the pelletmorphology relative to the filamentous morphology.

The amino acid sequence of Arsa-43 predicted from SEQ ID NO.:33, is setforth in SEQ ID NO.:34. FIG. 17 shows the BLASTP homology search resultsfor Arsa-43. The results show that the predicted amino acid sequence ofArsa-43, SEQ ID NO.:34 (top sequence), has 96% identity with the aminoacid sequence of the polyubiquitin protein from Aspergillus nidulans,SEQ ID NO.:35 (bottom sequence).

FIG. 18 shows the results of Northern blot analysis of the expression ofthe Arsa-10 gene in native A. niger corresponding to the cDNA partialsequence set forth in SEQ ID NO.:36. The results indicate that theArsa-43 gene is differentially expressed, with increased expression inthe pellet morphology relative to the filamentous morphology. Homologysearching is unable to detect any homology between SEQ ID NO.:36 andother polynucleotide sequences in the searched database

FIG. 19 shows the results of Northern blot analysis of the expression ofthe Arsa-27 gene in native A. niger corresponding to the cDNA sequenceset forth in SEQ ID NO.:37. The results indicate that the Arsa-43 geneis differentially expressed, with increased expression in the pelletmorphology relative to the filamentous morphology. Homology searching isunable to detect any homology between SEQ ID NO.:37 and otherpolynucleotide sequences in the searched database.

Referring to FIGS. 20 and 21, such show bar-chart comparison ofdifferential expression of various A. niger genes. FIG. 20 showstranscript levels for genes Balu-4. Brsa-25, Brsa-43, Brsa-47, Brsa-109and Brsa-118, which show increased expression in filamentous A. niger.FIG. 21 shows transcript levels for genes Arsa-7, Arsa-10, Arsa-27,A-37, Arsa-43, and A-90, which show increased expression in the pelletmorphology of A. niger.

Additional expression analysis is conducted utilizing cultures grown forup to 5 days post t=0 (as defined above). Referring to FIG. 22, suchshows the increased transcript levels for genes Balu-4, Balu-42,Brsa-25, Brsa-47, Brsa-109, and Brsa-118 in native A. niger grown infilamentous conditions (Panel B) as compared to transcript levels in A.niger grown in pellet conditions (Panel A). Referring to FIG. 23, suchshows the increased transcript levels for genes Arsa-7, A-37, Arsa-48and A-90 in native A. niger grown in pellet conditions (Panel A), ascompared to levels of the corresponding transcript in filamentouscultures (Panel B).

In particular embodiments, the present invention encompasses isolatedpolypeptide molecules comprising an amino acid sequence set forth in anyof SEQ ID NOs.:2, 5, 7, 9, 13, 17, 19, 23, 25, 27, 29 and 34, andfunctional equivalents thereof. For purposes of the present description,the term functional equivalent can refer to a truncated version or aconservatively substituted version of an amino acid sequence havingsubstantially equivalent functional properties and/or biologicalactivity relative to the non-truncated, non-substituted polypeptide. Aswill be understood by those skilled in the art, conventional methods canbe utilized for truncating or introducing conservative amino acidsubstitutions into the amino acid sequences set forth in the sequencelisting. Conventional methods are available that can be utilized forproducing of the isolated polypeptides of the present invention.

In addition to the isolated polynucleotide molecules discussed above,the present invention encompasses polynucleotides comprising alternativepolynucleotide sequences that encode the amino acid sequences set forthin SEQ ID NOs.:2, 5, 7, 9, 13, 17, 19, 23, 25, 27, 29 and 34, or thatencode functional equivalents of those amino acid sequences. Theinvention also encompasses amino acid sequences encoded by SEQ IDNOs.:36 and 37, and functional equivalents, and alternate polynucleotidesequences encoding the amino acid sequences encoded by SEQ ID NOs.:36and 37. As will be under stood by those skilled in the art, variousmodifications can be introduced into a polynucleotide sequence withoutaffecting the resulting amino acid sequence due to the degenerativenature of the genetic code.

Various recombinant polynucleotide constructs are encompassed by thepresent invention. In particular embodiments, a recombinantpolynucleotide construct according to the present invention can compriseany of the isolated polynucleotide sequences discussed above. All orpart of any of the polynucleotide sequences discussed herein can belinked to a promoter, preferably operably linked to a promoter. Operablelinkage of a polynucleotide to a promoter to form a recombinantpolynucleotide construct can allow expression of the polynucleotidesequence to be controlled by the promoter. Alternatively, a sequencecomplimentary to at least a part of a sequence set forth in any one ofSEQ ID NO.:1, 4, 6, 8, 12, 16, 18, 22, 24, 26, 28, 33, 36 and 37, can beutilized to form a recombinant polynucleotide, and can be incorporatedin antisense orientation.

In particular aspects, the complementary sequence can comprise a portionof complementary sequence of sufficient length to enable suppressionhybridization (discussed below). Although utilization of polynucleotidesequences of fewer than 30 nucleotides is contemplated, suppressionhybridization can typically involve utilization of one or morepolynucleotides having a length of greater than or equal to 30nucleotides. Accordingly, the invention encompasses polynucleotidesequences comprising a fragment of any of the sequences set forth in anyone of SEQ ID NO.:1, 4, 6, 8, 12, 16, 18, 22, 24, 26, 28, 33, 36 and 37,and complimentary fragments. Such fragments can preferably comprise alength of at least 30 nucleotides of the corresponding sequence, orcomplimentary sequence.

The invention also encompasses a vector comprising any of the isolatedpolynucleotide sequences discussed above. Vectors encompassed by thepresent invention are not limited to a particular type of vector and canbe, for example, a plasmid, a cosmid or a viral vector. Vectorsaccording to the present invention can be utilized for introducing intoa host cell one or more of the isolated polynucleotide moleculesdiscussed. The host cell is not limited to a particular cell type andcan be, for example, a bacterium, a fungus, or a higher-eukaryotic cell.Additionally, vectors encompassed by the present invention can becloning vectors, expression vectors and/or integration vectors.

The invention also encompasses a transformed host cell and cell cultureswhich have been transformed to comprise any of the isolatedpolynucleotide molecules discussed above. Conventional celltransformation techniques can be utilized for introduction of theisolated polynucleotide into a desired host cell.

The present invention encompasses methods for promoting a morphology ina fungus. A process for promoting a morphology in a fungus is describedwith reference to a flowchart in FIG. 24. At initial step 100, anisolated polynucleotide is provided. The isolated polynucleotide fromstep 100 can comprise any of the isolated polynucleotides discussedabove.

The isolated polynucleotide from step 100 can be used to form arecombinant polynucleotide in step 110. As discussed above, formation ofthe recombinant polynucleotide can comprise operably linking a promoterand the isolated polynucleotide sequence. Additionally, formation of arecombinant nucleotide step 110 can comprise formation of a vector whichcan be utilized to transform a fungus in step 120. Numerous fungi areavailable for utilization in transformation step 120. Preferably thefungus to be transformed is capable of exhibiting a filamentousmorphology and is additionally capable of exhibiting a pelletmorphology. Exemplary fungi for purposes of step 120 can be, forexample, any of the fungi discussed above with respect to source fungi.

After transformation step 120, a polypeptide encoded by the recombinantpolynucleotide can be expressed from the transformed fungus in step 130.The expression in step 130 can promote a particular morphology of thefungus. The particular morphology promoted by the expression can bedetermined by the sequence of the isolated polynucleotide provided instep 100. For example, a filamentous morphology can be promoted byproviding an isolated polynucleotide encoding a polypeptide comprisingan amino acid sequence set forth in any one of SEQ ID NOs.: 2, 5, 7, 9,13, 17, and 19, and functional equivalents thereof. Alternatively, apellet morphology can be promoted by providing an isolatedpolynucleotide in step 100 that encodes a polypeptide comprising anamino acid sequence set forth in any one of SEQ ID NOs.:23, 25, 27 29,and 34, or a functional equivalent thereof; or that encodes an aminoacid sequence encoded by polynucleotide SEQ ID NO.: 36 or 37, or afunctional equivalent thereof.

In an alternate embodiment of the present invention, a recombinantpolynucleotide comprising an antisense oriented complimentary sequence(discussed above) can be utilized for transformation step 120. In asuppression step 140, the RNA produced from transcription of theantisense DNA can form an RNA duplex (dsRNA) with the native mRNA andthereby promote RNA degradation and/or inhibit or block translation ofthe mRNA. Accordingly, recombinant antisense constructs introduced instep 120 can suppress or block expression of the complimentary gene topromote a desired morphology. For example, a polynucleotide constructcomprising, a sequence complimentary to a fragment or an entirety of anyof SEQ ID NOs.:1, 4, 6, 8, 12, 16 or 18 can be introduced in step 120.In step 140, the transcript produced from the antisense complimentarysequence can hybridize to mRNA transcribed from genes Balu-4, Balu-42,Brsa-25, Brsa-43, Brsa-47, Brsa-109 or Brsa-118, respectively, andinhibit or block production of the corresponding protein product.Suppression of one or more of Balu-4, Balu-42, Brsa-25, Brsa-43,Brsa-47, Brsa-109 or Brsa-118 by methods in accordance with the presentinvention can promote pellet morphology in the transformed host.Similarly, polynucleotides having one or more sequences complimentary toa fragment or an entirety of any of SEQ ID NOs.: 22, 24, 26, 28, 33, 36,and 37, can be introduced in step 120, can inhibit or block expressionof corresponding gene Arsa-7, Arsa-48, A-37, A-90, Arsa-43, Arsa-10 andArsa-27. Suppression of one or more of Arsa-7, Arsa-48, A-37, A-90,Arsa-43, Arsa-10 and Arsa-27 in step 140 by methods in accordance withthe present invention can promote filamentous morphology in thetransformed host.

Although the process shown in FIG. 24 was discussed in terms ofproviding a single isolated polynucleotide in step 100, it is to beunderstood that the invention encompasses providing two or more of theisolated polynucleotide sequences discussed above. Additionally, it isto be understood that isolated polynucleotide sequences can be providedin step 100 wherein at least one of the isolated polynucleotidesprovided can promote pellet morphology when expressed and at least oneother provided isolated polynucleotide can promote filamentousmorphology when expressed. By operably linking differing isolatedpolynucleotides to differing inducible promoters in step 110, and usingmultiple recombinant polynucleotides for transformation step 120, it canbe possible to selectively promote either the filamentous morphology orthe pellet morphology by inducing expression in step 130 or 140.

It can be advantageous to promote a particular morphology in a fungussince utilization of a particular fungus morphology can enhance abioprocess in a fungus culture. For example, utilization of a pelletform of a fungus can enhance various bioprocesses such as, for example,expressing hemicellulase, expressing cellulase, expressing lignase,converting biomass to alcohol, producing organic acids, producingglucoamylase, producing penicillin and producing lovastatin.Alternatively, utilization of filamentous fungal cultures can enhancebioprocesses such as fumaric acid production or peptic enzymeproduction.

The process shown in FIG. 24 can be utilized to produce a transformedfungus and to promote a pellet morphology in the transformed funguswhich can be utilized to enhance production of a desired product in aculture containing the transformed fungus relative to non-transformedfungus cultures under otherwise identical conditions. Alternatively, theprocess can be utilized to produce a transformed fungus and to promote afilament morphology in the transformed fungus. The promoted filamentmorphology can enhance production of a desired product in a culturecontaining the transformed fungus relative to non-transformed fungusculture under otherwise substantially identical conditions.

The invention also contemplates co-introduction of one or morepolynucleotides encoding one or more proteins of interest along with themorphology promoting constructs discussed above. The protein of interestcan be native to the host or can be from a different fungal ornon-fungal species. Where the protein(s) of interest have increasedexpression and/or activity in a first morphology relative to a secondmorphology, the morphology promoting construct co-introduced canpreferably promote the first morphology. A protein of interest may beone that can be collected from the culture or can be one that isinvolved in a bioprocess that produces a desired product or compound.

EXAMPLES Example 1 General Methods for DNA Isolation and FunctionalAnalysis.

Escherichia coli (E. coli) strains DH5α and JM109 are used as hosts forcloning experiments. Agrobacterium tumefaciens strain AGL0 is utilizedas host for binary vectors and transformation of A. niger.

For isolation of morphology associated genes by suppression subtractivehybridization (SSH), total RNA is isolated from A. niger according tothe modified acid phenol-guanidinium isothiocyanate-chloroformextraction method described by Chomczynski and Sacch (Anal. Biochem.162:156-159 (1987)). The SSH is performed utilizing the PCR-SELECT™ cDNAsubtraction kit (CLONTECH, Palo Alto Calif.) as described by themanufacturer, with the exception that the amount of amount of drivercDNA relative to tester utilized was tripled for each of the first andthe second hybridizations.

Morphology associated clones are identified by differential screening ofSSH cDNA libraries. Two oligonucleotides are designed against each newlyisolated clone sequence. Rapid amplification of cDNA and PCR (RACE-PCR)is performed to isolate the 5′-end and the 3′-end of each cDNA clone.

Fungal transformation is achieved utilizing the Bgl Il/Xba IpGpdA-hph-TtrpC fragment in pAN7-1 (Punt and van der Hondel, MethodsEnzymol. 216: 447-57 (1992)), inserted into binary vector pGA482 (An etal., Binary Vectors” in Plant Molecular Biology Manual, Gelvin andSchilperolands (1988), at pp A3/1-19). Introduction of constructs basedon pGA482 into Agrobacterium tumefaciens strain AGL0 is conductedutilizing the freeze-and-thaw method (Ebert et al., Proc. Natl. Acad.Sci., USA 84: 5745-5749 (1987)). Plasmids are isolated from thetransformed A. tumefaciens, are digested with various restrictionenzymes, and are analyzed utilizing agarose gel electrophoresis toconfirm transformation. Fungal transformations are performed asdescribed by Groot et al. (Nat. Biotechnol. 18: 839-42 (1998). At leastfifteen independently transformed fungi are selected and grown on agarminimum media containing 250 μg/ml of hygromycin, and 250 μg/mlcefotaxin for each transgenic event.

Example 2 Promoting a Morphology using Antisense Expression.

Individual transgene expression vectors are constructed to comprisepolynucleotide sequence complimentary to one the following: Balu-42 (SEQID No.: 4); Brsa-25 (SEQ ID No.:6); Brsa-118 (SEQ ID No.:18); Arsa-7(SEQ ID No.:22); A-37 (SEQ ID No.:26); and A-90 (SEQ ID No.:28). Thecomplimentary sequences are incorporated into the vectors in antisenseorientation under the control of A. nidulans phosphoglyceraldehydrogenase (gpdA) promoter and A. nidulans trpC terminator. Theconstructed vectors are independently introduced into A. niger utilizingAgrobacterium tumefaciens mediated transformation. Control A. niger isprepared by transformation with binary vector without incorporatedantisense sequence.

Referring to FIG. 25, such shows the promotion of the pellet morphologyin transgenic A. niger expressing antisense Balu-42, Brsa-25 andBrsa-118 (right), as compared to control A. niger cultured underidentical conditions. FIG. 26 shows the promotion of filamentousmorphology in transgenic A. niger expressing antisense Arsa-7, A-37 andA-90 (right), as compared to control A. niger cultured under identicalconditions.

Example 3 Morphology Enhanced Bio-production.

Transgenic A. niger comprising antisense complimentary Balu-42 (strain2805) or Brsa-118 (strain 2808) is prepared as described in Example 1.Multiple independently transformed cultures of each strain and multiplecontrol cultures (prepared as described above) were grown at 30° C. forabout 50 hours. Referring to FIG. 26, Panel A shows the citric acidproduction for individual cultures of transformed strains 2805 (Balu-42)and 2808 (Brsa-118), and for control A. niger. Panel B shows the averagecitric acid production for cultures of strains 2805 and 2808 relative tocontrol cultures.

The results indicate that the methods and sequences of the invention canbe utilized to promote morphology in fungi. The promotion of amorphology by methodology of the invention can be used for enhancingproduction of protein and/or enhancing a bioprocess utilizing transgenicfungi.

In compliance with the statute, the invention has been described inlanguage more or less specific as to structural and methodical features.It is to be understood, however, that the invention is not limited tothe specific features shown and described, since the means hereindisclosed comprise preferred forms of putting the invention into effect.The invention is, therefore, claimed in any of its forms ormodifications within the proper scope of the appended claimsappropriately interpreted in accordance with the doctrine ofequivalents.

1. A method of promoting a morphology in a fungus comprising: providinga recombinant polynucleotide comprising an antisense orientated sequencethat is complementary to a gene coding region that is differentiallyexpressed in a native fungus exhibiting a pellet morphology relative tosaid native fungus exhibiting a filament morphology wherein thecomplementary sequence is complementary to an entirety of any one of SEQID NOs.:1, 6, 8, 12, 16, 18, 22, 24, 26, 28, 33, 36 and 37; transformingAspergillus niger, transcribing the antisense oriented sequence toproduce a transcription product of sufficient length to hybridize to agene coding sequence transcription product to block translation; andsuppressing expression of the gene coding region utilizing transcriptionproducts produced by expression of the recombinant polynucleotide, thesuppression promoting a pellet or filament morphology capable of beingassumed by the fungi in its native form.
 2. A method of enhancing abioprocess utilizing a fungus, comprising: producing a transformedfungus by transforming Aspergillus niger with a recombinantpolynucleotide molecule comprising a polynucleotide sequencecomplementary to the entirety any one of SEQ ID NOs.: 1, 6, 8, 12, 16,18, 22, 24, 26, 28, 33, 36 and 37, linked operably to a promoter, thepolynucleotide sequence being in antisense orientation; transcribing thepolynucleotide sequence to produce polynucleotide transcripts; andhybridizing the transcripts to mRNA to suppress gene expression andpromote pellet or filament morphology, the promoted morphology enhancinga bioprocess relative to the bioprocess utilizing an opposing filamentor pellet morphology of the transformed fungus.
 3. The method of claim 2wherein the promoted morphology is filament morphology.
 4. The method ofclaim 2 wherein the promoted morphology is pellet morphology.